Solving a Sticky Problem: Understanding Enzyme Binding to Lignocellulosic Biomass during Biofuel Production

PI：Whitehead，时间建议编号：1236120机构：密歇根州立大学标题：解决一个棘手的问题：了解在生物燃料生产过程中酶与木质纤维生物质的结合生物介导性地将纤维生物质转化为有用的燃料和化学品是实现能源可持续发展的一条有希望的途径。这条道路的一个关键障碍是将生物质分解为可发酵糖所需的纤维素酶的成本。在目前纤维素酶到底物的负载下，木质纤维素生产的每加仑燃料的成本仍然比降解淀粉所需的酶高出至少一个数量级，即使酶的生产成本可以像大豆蛋白一样低。减少酶需要量的一种策略是在连续的过程中回收纤维素酶，但生物质糖化过程中的酶失活严重限制了这一方法。Pi？S最近的发现表明，对于大多数前处理化学品和生物质来源，酶的失活是通过与残留木质素的非生产性相互作用(即纤维素酶吸附到疏水的木质素表面加速其变性)而发生的。PI假设最小化纤维素酶和木质素聚合物之间的平均作用力势将减少两者之间的表面吸附，从而最小化酶的失活。因此，本项目的主要目的是验证这样的假设，即纤维素酶表面化学的选择性变化可以减少由于蛋白质表面对木质素的吸附减少而导致的木质素介导的失活。这一假设将通过使用计算蛋白质设计来验证，以确定导致不同酶表面电位的突变，预测这些突变将使酶-木质素吸附最小化，然后在主要微生物里氏木霉的两种典型纤维素酶中对这些突变进行遗传编程。大量(约1000个)纤维素酶变异体将被重组表达，并在纤维素底物上进行溶解性和活性的筛选。存活的变异体将接受结构保留、溶解性和稳定性的测试。不同表面化学和微观结构的天然木质素和合成木质素的变种的吸附等温线将被记录下来。PI将对纤维素酶催化域、碳水化合物结合模块和全长纤维素酶进行测试。该项目将提供对蛋白质表面吸附效应的基本定量理解，对利用木质纤维素生物质生产商业规模的生物能源具有重要意义。更广泛地说，这项工作将为确定控制酶-表面相互作用的关键参数提供数据集，这些参数对不同的应用至关重要，例如分离单壁碳纳米管、洗涤剂、医用生物材料和固定化生物催化剂。该项目将吸引来自不同跨学科项目和代表性不足群体的K-12、本科生和研究生水平的学生，以拓宽他们对工业生物技术在以环境可持续的方式缓解与生物能源相关的问题方面的作用的理解。该项目将利用在五大湖生物能源研究中心建立的外展教育计划，为密歇根州中部学校社区内的科学教师创建与生物能源相关的教育材料。

PI: Whitehead, TimProposal Number: 1236120Institution: Michigan State UniversityTitle: Solving a Sticky Problem: Understanding Enzyme Binding to Lignocellulosic Biomass during Biofuel ProductionBiological-mediated conversion of cellulosic biomass to useful fuels and chemicals is a promising avenue towards energy sustainability. A critical impediment for this avenue is the cost of the cellulase enzymes needed to deconstruct biomass to fermentable sugars. At the current cellulase-to-substrate loading the cost per gallon of fuel produced from lignocellulose would still be at least an order of magnitude higher than the enzymes required to degrade starch, even if the enzymes could be produced as cheaply as soy protein. One strategy to reduce enzyme requirements is to recycle the cellulases in a continuous process, but enzyme inactivation during biomass saccharification severely limits this approach. PI?s recent findings show that enzyme inactivation occurs through non-productive interactions with residual lignin (i.e. the cellulases adsorb to hydrophobic lignin surfaces hastening their denaturation) for most pretreatment chemistries and biomass sources.The PI hypothesizes that minimizing the potential of mean force between the cellulases and lignin polymer will reduce surface adsorption between the two, hence minimizing enzyme inactivation. Thus, the primary objective of this project is to test the hypothesis that selective variation of cellulase surface chemistry can decrease lignin-mediated inactivation due to reduced protein surface adsorption to lignin. The hypothesis will be tested by using computational protein design to identify mutations that result in different enzyme surface potentials predicted to minimize enzyme-lignin adsorption, and then genetically program these mutations in two representative cellulases from the workhorse microbe Trichoderma reesei. A large number (ca. 1000) of cellulase variants will be recombinantly expressed and screened for solubility and activity on cellulosic substrates. Surviving variants will be tested for retention of structure, solubility, and stability. Adsorption isotherms will be recorded for the variants for natural and synthetic lignin of varying surface chemistry and microstructure. The PI will test this hypothesis for cellulase catalytic domains, carbohydrate binding modules, and the full-length cellulases.The project will provide fundamental quantitative understanding of protein-surface adsorption effects with significant implications for commercial-scale bioenergy production using lignocellulosic biomass. More generally, this work will supply datasets for identifying critical parameters governing enzyme-surface interactions crucial for disparate applications such as separations of single wall carbon nanotubes, laundry detergents, biomaterials for medical uses and immobilized biocatalysts.This project will engage students from K-12, undergraduate, and graduate levels from various interdisciplinary programs and under-represented groups to broaden their understanding of the role of industrial biotechnology in mitigating bioenergy related problems in an environmentally sustainable manner. The project will utilize the established outreach educational programs at the Great Lakes Bioenergy Research Center to create bioenergy-relevant educational materials for science teachers within the greater mid-Michigan school community.